The multi-catalytic proteasome is the ubiquitous proteinase found in cells throughout the plant and animal kingdoms that is responsible for the ubiquitin-dependent degradation of intracellular proteins. Thousands of copies are found in all cells, in both the cytoplasm and the nucleus, which constitute up to 3% of all cellular protein content. Proteasomes serve multiple intracellular functions, including the degradation of damaged proteins and the modulation of many regulatory proteins that affect inflammatory processes, viral shedding, the cell cycle, growth, and differentiation, to name but a few [Cell 1994, 79, 13-21; Nat. Rev. Mol. Cell Biol. 2005, 6, 79-87; Semin. Oncol. 2004, 31, 3-9; Chem. Biol. 2001, 8, 739-758].
The ubiquitin-proteasome pathway (UPP), also known as the ubiquitin-proteasome system (UPS), regulates the degradation of intracellular proteins with specificity as to target, time and space. The pathway plays a central role in recognizing and degrading misfolded and abnormal proteins in most mammalian cells [Nature 2000, 404, 770-774]. Such a process is very important in maintaining the biological homeostasis and regulation of different cellular processes such as but not limited to cell differentiation, cell cycle control, antigen processing and hormone metabolism [EMBO J. 1998, 17, 7151-7160; Chem. Biol. 2001, 8, 739-758]. In this pathway, the 26S proteasome is the main proteolytic component, which is found in all eukaryotic cells and is made up of the cylinder-shaped multi-catalytic proteinase complex (MPC) 20S proteasome and two regulatory particle (RP) 19S proteasomes. The 19S proteasome located at each end of the 20S proteasome is made up of 18 subunits, and controls the recognition, unfolding, and translocation of protein substrates into the lumen of the 20S proteasome [Annu. Rev. Biochem. 1999, 68, 1015-1068].
X-ray crystallography of the 26S proteasome revealed that the 20S proteasome is composed of 28 protein subunits arranged in four stack rings, with each ring made up of seven α- and β-type subunits, following an α1-7β1-7 stoichiometry [Science 1995, 268, 533-539; Nature (London) 1997, 386, 463-467]. The two outer chambers are formed by a subunits, while the central chamber, containing the proteolytic active sites, is made up of β subunits. Three of the 14 β subunits are responsible for the post-glutamyl peptide hydrolysis activity (PGPH, attributed to (β1), trypsin-like activity (T-L, (β2), and chymotripsin-like activity (CT-L, β5), respectively, and all these three active subunits hydrolyze the amide bond of protein substrates with the hydrophilic γ-hydroxyl group of the N-terminal threonine (Oγ-Thr1).
Rising interest in the mechanism and function of the proteasomes and the ubiquitin system revealed that it is hard to find any aspect of the cellular metabolic network that is not directly or indirectly affected by the degradation system. This includes, for example the cell cycle, the “quality control” of newly synthesized proteins (ERAD: Endoplasmic Reticulum Associated Protein Degradation), transcription factor regulation, gene expression, cell differentiation and immune response as well as pathologic processes such as cancer, neurodegenerative diseases, lipofuscin formation, diabetes, atherosclerosis, inflammatory processes and cataract formation in addition to the aging process and the degradation of oxidized proteins in order to maintain cell homeostasis. But this seems to be only a small aspect of the general view. The various regulator proteins that are able to change the rate or specificity of proteolysis, fitting it out for highly specialized tasks, or the precise regulation of the half-life of cellular proteins by ubiquitin-mediated degradation shape the proteasome and the ubiquitin-proteasome system into a useful part of cellular function in the three kingdoms of bacteria, plants and animals.
Cancer is a leading cause of death worldwide. Despite significant efforts to find new approaches for treating cancer, the primary treatment options remain surgery, chemotherapy and radiation therapy, either alone or in combination. Surgery and radiation therapy, however, are generally useful only for fairly defined types of cancer, and are of limited use for treating patients with disseminated disease. Chemotherapy is a method that is useful in treating patients with metastatic cancers or diffuse cancers such as leukemias. However, although chemotherapy can provide a therapeutic benefit, it often fails to result in cure of the disease due to the patient's cancer cells becoming resistant to the chemotherapeutic agent. Therefore, a need exists for additional chemotherapeutics to treat cancer.
The concept of proteasome inhibition as a therapeutic approach in cancer is known. The first-in-class inhibitor bortezomib is a potent, selective, and reversible proteasome inhibitor which targets the 26S proteasome complex and inhibits its function. Proteasomal degradation of misfolded or damaged proteins proceeds by recognition of poly-ubiquitinated proteins by the 19S regulatory subunit of the 26S protease, and subsequent hydrolysis to small polypeptides.
The successful development of bortezomib for treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, has shown proteasome inhibition to be a useful therapeutic strategy [Nat. Rev. Cancer 2004, 4, 349-360; Bioorg. Med. Chem. Lett. 1998, 8, 333-338; J. Clin. Oncol. 2002, 20, 4420-4427; N. Engl. J. Med. 2003, 348, 2609-2617; N. Engl. J. Med. 2005, 352, 2487-2498; J. Clin. Oncol. 2007, 25, 3892-3901]. Bortezomib primarily inhibits chymotryptic, without altering tryptic or caspase-like, proteasome activity. Bortezomib has pleiotropic effects on multiple myeloma biology by targeting a) cell-cycle regulatory proteins; b) the unfolded protein response (UPR) pathway via modulating the transcriptional activity of plasma cell differentiation factor X-box binding protein-I (XBP-I); c) p53-mediated apoptosis/MDM2; d) DNA repair mechanisms; and e) classical stress-response pathways via both intrinsic (caspase-9 mediated) and extrinsic (caspase-3 mediated) cell death cascades. Specifically, bortezomib activates c-Jun N-terminal kinase (JNK), which triggers mitochondrial apoptotic signalling: release of cytochrome-c (cyto-c) and second mitochondrial activator of caspases (Smac) from mitochondria to cytosol, followed by activation of caspase-9 and caspase-3.
Although bortezomib has shown clinical success, a significant fraction of patients relapse or are refractory to treatment [J. Clin. Oncol. 2005, 23, 676-684; J. Clin. Oncol. 2005, 23, 667-675]. Additionally, dose-limiting toxicities (DLT), including a painful peripheral neuropathy and thrombocytopenia, have been reported [J. Clin. Oncol. 2006, 24, 3113-3120; Blood 2005, 106, 3777-3784]. To date, it is unclear whether these toxicities can be attributed to off-target effects because bortezomib inhibits other enzymes such as serine proteases.
A recently reported structural analogue of the microbial natural product epoxomicin, known as carfilzomib (also called PR-171) was initially identified for its antitumor activity and subsequently shown to be a potent inhibitor of the proteasome [Cancer Res. 2007, 67, 6383-6391; Curr. Opin. Drug Discovery 2008, 11, 616-625; J. Am. Chem. Soc. 2000, 122, 1237-1238; J. Antibiot. (Tokyo) 1992, 45, 1746-1752; Bioorg. Med. Chem. Lett. 1999, 9, 2283-2288; Cancer Res. 1999, 59, 2798-2801; Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10403-10408]. Carfilzomib selectively inhibits the CT-L activity of the 20S proteasome with minimal cross reactivity to other protease classes.
Preclinical studies and phase I clinical studies demonstrated that consecutive daily dosing schedules with carfilzomib are both well-tolerated and promote antitumor activity in hematologic malignancies, including patients previously treated with bortezomib [Blood 2007, 110, 3281-3290; Br. J. Hamaetol. 2007, 136, 814-828; Blood 2007, 110, 409; Blood 2007, 110, 411]. Carfilzomib is currently being evaluated in phase I and phase II clinical trials in multiple myeloma, non-Hodgkin's lymphoma, and solid tumors.
Clinical responses to known proteasome inhibitor therapies require frequent dosing (e.g., twice per week) and prolonged treatment. For example, both bortezomib and carfilzomib are administered intravenously (iv) on biweekly or more frequent dosing schedules with a treatment that can extend for over 6 months. Therefore, the development of orally bioavailable proteasome inhibitors that would allow for dosing flexibility and improve patient convenience is desirable.
Proteasome inhibitor-based therapeutics are useful in other diseases beyond clinical oncology. In addition to its role in cancer therapy, the proteasome is linked to the production of the majority of the class I antigens [Nature 1992, 357, 375-379]. Therefore excessive inhibition of the proteasome might increase the chance of viral infections. For example, it was reported that replication of the HIV-1 virus could be limited by the degradative actions of the proteasome and that the proteasome inhibitor, MG-132 or lactacystin, enhanced the ability of the virus to replicate [J. Virol. 1998, 72, 3845-3850]. In contrast, a number of recent publications have suggested that the ubiquitin-proteasome pathway has a useful role in the processing of retroviral assembly, maturation, and budding [Proc. Natl. Acad. Soc. USA 2000, 97, 13069-13074; Proc. Natl. Acad. Sci. USA 2000, 97, 13057-13062; Proc. Natl. Acad. Sci. USA 2000, 97, 13063-13068]. Proteasome inhibition also interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2, and ubiquitination is required for retroviral release. Hence, proteasome inhibitors can be useful for the treatment of HIV and other viral infections.
Proteasome inhibition also has clinical potential for treatment of inflammatory and autoimmune diseases through multiple pathways, including MHC-mediated antigen presentation, cytokine and cell cycle regulation, and apoptosis [J. Rheumatol. 2005, 32, 1192-119]. In inflammatory arthritis, it was shown that NF-κB regulates multiple critical cytokines involved in the pathogenesis of rheumatoid arthritis (RA) [Arthritis Rheum. 2004, 50, 2381-2386; Arthritis Rheum. 2004, 50, 3541-3548]. In the peptoglycan/polysaccharide-induced inflammatory arthritis model, a proteasome inhibitor improved the arthritis score by suppressing the activation of NF-κB, reducing the expression of cell adhesion molecules and IL-6. In addition, proteasome inhibition may regulate the development of inflammatory arthritis by controlling angiogenesis [J. Mol. Med. 2003, 81, 235-245].
Psoriasis is one of the prototypical T cell-mediated diseases, and its development is related to the activation of NF-κB. Administration of a proteasome inhibitor has been reported to reduce the size of psoriatic lesions in human skin explants grafted onto mice. The treatment also resulted in reduced super antigen-mediated T-cell activation, attenuated cell adhesion molecule expression and decreased expression of T-cell activation markers that were significantly elevated during the disease process [J. Clin. Invest. 2002, 109, 671-679].
In addition, other studies showed oral proteasome inhibition by bortezomib significantly limited overall inflammation, reduced the activation of NF-κB, lowered cell adhesion molecule expression, inhibited nitric oxide synthase activity, attenuated the circulating levels of IL-6, reduced the arthritic index and swelling observed in the joints of the animals, and improved the histologic appearance of the joints compared with vehicle-treated animals [Carcinogenesis 2000, 2, 505-515].
A link between proteasome inhibition, allergy and asthma has also been shown. Abnormal activation of type 2 helper T cells (Th2) results in asthmatic and allergic symptoms [Nat. Immunol. 2002, 3, 715-720]. E3 ubiquitin ligase Itch plays a useful role in maintaining immune tolerance mediated through Th2 cells both in vitro and in vivo. Itch deficient mice failed to block the development of airway inflammation in an allergic model [J. Clin. Invest. 2006, 116, 1117-1126]. Consistent with these findings, useful therapeutic effects were observed in a rodent model of allergen-induced asthma [J. Allergy Clin. Immunol. 1999, 104, 294-300].
Other inflammatory and autoimmune diseases have been linked to the ubiquitin-proteasome system (UPS), such as seronegative spondyloarthropathies (SpA) which are a group of diseases characterized by, but not limited to, axial joint inflammation. Ankylosing spondylitis (AS) is the prototypical SpA. Most patients with AS carry the MHC class I HLA-B27 gene, and therefore much research effort has been directed at understanding the role of this gene in the disease pathogenesis. There has also been interest focused on determining the origin and nature of the peptides being presented by HLA-B27 and the cell surface expression of misfolded HLA-B27, two areas in which the UPS is known to play a role.
The UPS is involved in the regulation or induction of apoptosis. Apoptosis has been implicated in both experimental models and clinical systemic lupus erythematosus (SLE). In mature, activated lymphocytes, the proteasome inhibitor lactacystin induces DNA fragmentation and apoptosis in a dose-dependent fashion, indicating that proteasome suppresses apoptosis in these cells. Altered clearance of auto antigens is thought to allow for targeting by the immune system and the development of autoimmunity. The involvement of UPS in regulating the levels of Ku70 and other autoantigens has been reported [J. Biol. Chem. 1998, 273, 31068-31074; J. Cell. Sci. 1994, 107 (Pt 11), 3223-3233; Exp. Cell. Res. 2006, 312, 488-499].
Proteasome inhibition has also been linked to heart disease. Evidence continues to emerge to support a hypothesis that proteasome functional insufficiency represents a common pathological phenomenon in a large subset of heart disease, compromises protein quality control in heart muscle cells, and thereby acts as a major pathogenic factor promoting the progression of the subset of heart disease to congestive heart failure. This front is represented by the studies on the UPS in cardiac proteinopathy, which have taken advantage of a transgenic mouse model expressing a fluorescence reporter for UPS proteolytic function.
In addition, pharmacological inhibition of the proteasome has been explored experimentally as a potential therapeutic strategy to intervene on some forms of heart disease, such as pressure-overload cardiac hypertrophy, viral myocarditis, and myocardial ischemic injury [Biochimica et Biophysica Acta—Gene Regulatory Mechanisms, 1799(9), 2010, 597-668]. Furthermore, initial reports on the effects of proteasome inhibitors in cardiovascular diseases, indicate that proteasome inhibition might be a useful therapeutic strategy for the reduction of the proliferative phenomena of the progression stage of atherogenesis [Cardiovasc. Res. 2004, 61, 11-21]. Recent data on the improvement of endothelium-dependent vasorelaxation in vitro, correlating with an increase in endothelial nitric oxide synthase (eNOS) expression, suggest a therapeutic potential of proteasome inhibition in the early stages of atherosclerosis [FASEB 2004, 18, 272-279].
Proteasome inhibitors have been shown to exert a substantial anti-inflammatory effect, which was attributed to a reduction in the activity of the factor NF-κB [Cardiovasc. Res. 2004, 61, 11-21]. As the pathogenesis of cardiovascular events in diabetic patients involves inflammation, the use of proteasome inhibitors may be a useful therapy. In addition to epidemiological evidence for the role of inflammation in diabetes-associated cardiovascular events, clinical studies of patients on cardio-protective drug regimens have revealed that many of the pharmacotherapies mediate their benefits, at least in part, through anti-inflammatory activities. This is the case for one class of drugs that improves adipose tissue physiology and insulin sensitivity, the peroxisome proliferator-activated receptor-γ (PPARγ) agonists [Arterioscier. Thromb. Vasc. Biol. 2002, 22, 717-726]. For example, the PPARγ agonist rosiglitazone, reducing inflammation, may prevent plaque progression to an unstable phenotype in diabetic patients with asymptomatic carotid stenosis, enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis [Diabetes 2006, 55, 622-632].
The anti-inflammatory effects of glitazones are felt to be mediated partly by their beneficial effects on glycemia, but there is also evidence that glitazones may directly modulate inflammation via transcription factors such as NF-κB [Arterioscler. Thromb. Vasc. Biol. 2002, 22, 717-726]. In line with this, recent data have shown an inhibitory effect of rosiglitazone on ubiquitin-proteasome activity in diabetic lesions [Diabetes 2006, 55, 622-632]. At the same level of blood glucose levels, diabetic patients treated with rosiglitazone had the lowest level of ubiquitin and proteasome 20S activity, plaque inflammatory cells, cytokines, oxidative stress and MMP-9 associated with the highest content of plaque interstitial collagen. Patients assigned to rosiglitazone had lesser plaque progression to an unstable phenotype compared with patients assigned to placebo.
For aspirin and statins, two of the most successful drugs in treatment of cardiovascular diseases, a proteasome inhibitory effect has been described [Mol. Pharmacol. 2002, 62, 1515-1521].
Drugs that modulate the proteasomal degradation of proteins could be useful agents for the treatment of insulin-resistant and type-2 diabetes, and pharmacological therapies targeting UPS activity may be useful in the treatment of vascular biology disorders associated with diabetes [Cardiovascular Diabetology 2007, 6:35, 1-9].
The ubiquitin-proteasome system is also believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase-3) and downstream (tri-peptidyl-peptidase II and amino-peptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few proteins that seem necessary for muscle wasting i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 (muscle-specific RING ubiquitin-protein ligases) proteins. The characterization of their signaling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients [Essays Biochem. 2005, 41, 173-86].
The UPS has also been linked to the development of human obesity. For example, it was shown that there is a possible correlation between plasma ubiquitin, 26S proteasome levels, and obesity. The body mass index (BMI), plasma ubiquitin levels, and 26S proteasome activity levels were determined and statistically analyzed. Comparison of the immunoglobulin among the underweight, normal weight, and overweight groups demonstrated that plasma ubiquitin is significantly decreased in obese individuals versus normal controls, and plasma ubiquitin levels were found to be inversely correlated with the BMI. In addition, there was an inverse relationship between 20S proteasome levels in red blood cells and BMI, whereas 26S proteasome activity was found to be dependent quantitatively to S5a in erythrocytes. Furthermore, immunoglobulin is significantly decreased in overweight individuals versus normal controls [Metabolism 2009, 58(11), 1643-8].
A wide variety of preclinical and early clinical studies have been performed to test the potential usefulness of proteasome inhibitors for the treatment of neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's (PD) diseases. These CNS disorders are characterized by a selective loss of neurons in specific, but different, regions of the brain, and the result is often a disruption to motor, sensory or cognitive systems, resulting in severe disability of the patient. The pathological characteristic of many neurodegenerative diseases is the presence of distinctive ubiquitin-positive, intra- or extracellular inclusion bodies in affected regions of the brain. In general, these inclusions are made up of insoluble, unfolded, ubiquitylated polypeptides that fail to be targeted and degraded by the 26S proteasome [J. Pathol. 1988, 155, 9-15; Neuron 2001, 29, 15-32]. Their apparent stability may, in part, be due to decreased levels of 26S proteasomal activity that is associated with increasing age [Ann. N.Y. Acad. Sci. 2001, 928, 54-64].
Proteins associated with the UPS are now known to play either a direct or indirect role in familial forms of neurodegenerative disease and, in particular, PD. UPS-mediated post-translational modification and degradation of proteins is useful for most cellular processes such as cell cycling, DNA repair, cell signaling, gene transcription and apoptosis. Historically, it was recognized that the UPS is the major route by which proteins are selected for temporal and spatial degradation in eukaryotic organisms [Cell 2004, 116, 181-190; Nat. Rev. Mol. Cell Biol. 2003, 4, 192-201]. The key constituents of the inclusions associated with neurodegenerative disorders are mis-folded proteins. The major causes of protein mis-folding and subsequent loss of function are mis-sense mutations, modifications or posttranslational damage of proteins, or expansion of amino acid repeats as is observed in polyglutamine (polyQ) disorders such as Huntington's disease (HD).
Of all the neurodegenerative diseases, PD is most closely associated with aberrant protein processing via the UPS. Indeed, of the known proteins associated with hereditary forms of PD, Parkin and UCH-L1 are components of the UPS, whereas modified and/or mutant α-Synuclein and DJ-1 are degraded by the system [Nature 1998, 392, 605-608; Nature 1998, 395, 451-452; J. Biol. Chem. 2003, 278, 36588-36595].
A wide variety of preclinical and early clinical studies have been performed to test the potential usefulness of proteasome inhibitors for the treatment of Alzheimer's disease [J. Neurochem. 1999, 72, 255-261], amyotrophic lateral sclerosis [J. Neurol. Sci. 1996, 139, 15-20], autoimmune thyroid disease [Tissue Antigens. 1997, 50, 153-163], cachexia [N. Engl. J. Med. 1996, 335, 1897-1905; Am. J. Physiol. 1999, 277, 332-341], Crohn's disease [J. Pharmacol. Exp. Ther. 1997, 282, 1615-1622], Hepatitis B [Oncogene, 1998, 16, 2051-2063], inflammatory bowel disease [Inflamm. Bowel Dis. 1996, 2, 133-147], sepsis [Ann. Surg. 1997, 225, 307-316], systemic lupus erythematosus [J. Exp. Med. 1996, 10, 1313-1318], and transplantation rejection and related immunology [Drug Discov. Today 1999, 4, 63-70; Transplantation 2001, 72, 196-202].
The ubiquitin-proteasome system is also believed to play roles in the pathogenesis of eye diseases. Accumulation of the cytotoxic abnormal proteins in eye tissues is etiologically associated with many age-related eye diseases such as retina degeneration, cataract, and certain types of glaucoma. Age- or stress-induced impairment or overburdening of the UPP appears to contribute to the accumulation of abnormal proteins in eye tissues. Cell cycle and signal transduction are regulated by the conditional UPP-dependent degradation of the regulators of these processes. Impairment or overburdening of the UPP could also result in dysregulation of cell cycle control and signal transduction. The consequences of the improper cell cycle and signal transduction include defects in ocular development, wound healing, angiogenesis, or inflammatory responses. Methods that enhance or preserve UPP function or reduce its burden may be useful strategies for preventing age-related eye diseases [Pro. Mol. Biol. & Trans. Sc., Vol. 109, 2012, 347-396].
The search for subunit selective inhibitors is predominantly conducted by either screening of natural products [Bioorg. Med. Chem. Lett. 1999, 9, 3335-3340], rational design [Chem. Biol. 2009, 16, 1278-1289], or compound library building [Proc. Natl. Acad. Sci. USA 2001, 98, 2967-2972; Org. Biomol. Chem. 2007, 5, 1416-1426]. It was noted that in these studies the effect of fluorine functionality in proteasome inhibitors is relatively uncharted [Bioorg. Med. Chem. Lett. 2009, 19, 83-86].
The epoxomicin analog PR-047 was recently reported to be an orally-bioavailable candidate that displayed moderate to poor metabolic properties [J. Med. Chem. 2009, 52, 3028-3038]. While not wishing to be limited by theory, this poor metabolic property is thought to be due to the methoxy groups in the serine (OMe) side-chains undergoing demethylation to the 0-desmethyl metabolite. A need therefore exists to find a route to block this demethylation pathway to give compounds having useful clinical profile.